During the past 35 years this laboratory has been studying the function of polyisoprenol-linked intermediates in glycan synthesis in prokaryotes and eukaryotes. A long-standing interest in the topological issues involved in the biosynthesis of extracellular saccharides has led us to focus on the proteins that mediate the transbilayer movements of the lipid-linked intermediates utilized as glycosyl donors in these pathways. The current proposal is aimed at identifying the proteins that mediate the transbilayer movement of dimannosyldiacylglycerol and mannosylphosphoryldolichol, two mannolipid intermediates in the assembly of the lipomannan of Micrococcus luteus. These studies will provide important clues to the identification of related proteins functioning in protein N-glycosylation, C- and O-mannosylation of proteins and biosynthesis of glycosylphosphatidylinositol anchors in humans. The discovery of genes encoding membrane proteins functioning as flippases in these assembly processes will be valuable in the diagnosis of potential genetic errors referred to as Congenital Disorders in Glycosylation (CDGs). PUBLIC HEALTH RELEVANCE: This application proposes studies designed to identify the membrane proteins (flippases) in Micrococcus luteus that mediate the transbilayer movements of dimannosyl-diacylglycerol and mannosylphosphorylundecaprenol, two mannolipid intermediates in bacterial lipomannan synthesis. These studies on micrococcal lipomannan assembly will provide important new clues to the identification of related membrane proteins functioning in protein N-glycosylation, C- and O-mannosylation of proteins, and the biosynthesis of glycosylphosphatidylinositol (GPI) anchors in humans. Since N-linked oligosaccharides play a key role in the proper folding and intracellular routing of important membrane glycoproteins, e.g. rhodopsin, voltage-sensitive sodium channels and the insulin receptor, the identification of the genes encoding mammalian flippases is relevant to the diagnosis of potential genetic errors referred to as Congenital Disorders in Glycosylation (CDGs).